Julia McKenzie writes:
> ... I am using PCR to amplify
> mouse DNA using a variety of SSLP's obtained from the Whitehead
> Institute. [Doing an LOH assay; was working]...
> Now what I am seeing on these gels is one band only.
> Bizarre! It appears that the primers are preferentially annealing to
> and amplifying one allele. However, when I run the 2 genomic DNA
> controls I get amplification of one of them, the same one that
> amplified in the tumour DNA samples but there is no amplification of
> the other DNA control. What am I to make of this? Has something
> happened to the DNA? Should I try a hot start?
I always recommend hot start; but I don't see an obvious relation to your
problem. If there is a point mutation under one of the
primers linked to the allele that now doesn't amplify, it may be
that your stringency has just creeped up somehow. So you might
try dropping the annealing temp. about 5 C. Did I understand
correctly that this has happened for more than one primer pair?
If so, then it's hard to believe that there's a mutation under
serveral different primers. Did I understand that you have a PCR
mix that has been in the freezer a long time and you keep drawing
aliquots from it. If so, make up a fresh one. Your result with
the one control not amplifying is critical. Otherwise, I'd guess
that the PCR is actually working and instead you've lost resolution
in your gel system. So repeat this control and make sure you're not being
misled by an unrelated false result.
Sounds like a difficult puzzle.
Good luck.
Steve Hardies, Assoc. Prof. of Biochemistry, Univ. of Texas HSC at San Antonio
Hardies at uthscsa.edu